Not applicable.
The invention relates to monoclonal antibodies specifically directed against mutated transmembrane E-cadherin and useful for the detection and therapy of gastric carcinomas.
Introduction
Statistical evaluations of the causes of death show malignant tumors to be in a front position worldwide. Among these tumors the gastric carcinoma internationally takes the second position of tumors resulting in death. Several genetic alterations have been reported in association with the gastric carcinoma including microsatellite instabilities and alterations of the genes p53, APC DCC (Tahara E: Genetic alterations in human gastrointestinal cancers. Cancer 1995; 75:1410-1417). Histomorphologically, two types of gastric carcinoma can be distinguished (Laurxc3xa9n P: The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type of carcinoma. Acta Pathol. Microbiol. Scand. 1965; 64:31-49): one type are the intestinal carcinomas having a glandular differentiation, the other type consists of diffuse carcinomas with disrupted tissue architecture. So far, the genetic basis for this bipartite development and morphology has not been clarified. Possibly, alterations in the E-cadherin molecule may be of importance in this respect. E-cadherin is a homophilic transmembrane cellular adhesion molecule playing a key role in the interaction of epithelial tissues. Initial molecular-biological studies of the E-cadherin gene comprising 16 exons indicated that mutations may contribute to the morphology and growth type of gastric carcinomas (Becker K-F, Atkinson M J, Reich U, Becker I, Nekarda H, Siewert J R, Hxc3x6fler H: E-cadherin gene mutations provide clues to diffuse type gastric carcinomas. Cancer Res. 1994; 54(14):3845-3852).
Basically, alterations of malignant tumors are of interest for the explanation of certain biological patterns of behaviour; these phenomenons may be used as a specific characteristic feature and, therefore, as a tumor marker. These characteristics include cellular products and also typical properties of the cell surface which can be assessed in a direct or indirect manner. To date, the isolation of a surface antigen or cellular product restricted solely to tumor cells has not been successful. Up to now the increased occurrence of certain cellular properties (surface antigen, intracellular proteins, secreted cellular products) in the body relative to normal tissue has been used as the basis for diagnosis and therapy of malignant diseases.
State of the use of E-cadherin in diagnosis and therapy
The first publications reported that E-cadherinxe2x80x94detected by immunohistochemistry using specific antibodiesxe2x80x94showed an altered expression pattern in tumor cells. The immunoreactivity of E-cadherin was partly reduced in or absent from tumor tissue (see Table 1). Other authors thought this fact to be the reason for a decreased production (xe2x80x9cdownregulationxe2x80x9d) of the protein in the tumor (cf. Birchmeier, DE-A-41 10 405 A1).
References for Table 1.
1 Shino Y, Watanabe A, Yamada Y, Tanase M, Yamada T, Matsuda M, Yamashita J, Tatsumi M, Miwa T, Nakano H: Clinicopathologic evaluation of immunohistochemical E-cadherin Expression in human gastric carcinomas. Cancer 1995; 76:2193-2201.
2 Brito M J, Jacinto L, Jankowski J, Pignatelli M, Filipe MI: E-cadherin (cell adhesion molecule) in gastric carcinoma. Path. Res. Pract. 1995; 191:628 [Abstract].
3 Matsui S, Shiozaki H, Inoue M, Tamura S, Doki Y, Kadowaki T, Iwazawa T, Shimaya K, Nagafuchi A, Tsukita S, Mori T: Immunohistochemical evaluation of alpha-catenin expression in human gastric cancer. Virchows Archiv 1994; 424:375-381.
4 Mayer B, Johnson J P, Leitl F, Jauch K W, Heiss M M, Schildberg F W, Birchmeier W, Funke I: E-cadherin expression in primary and metastatic gastric cancer: down-regulation correlates with cellular dedifferentiation and glandular disintegration. Cancer Res. 1993; 53:1690-1695.
5 Shimoyama Y, Hirohashi S: Expression of E- and P-cadherin in gastric carcinomas. Cancer Res. 1991; 51(8):2185-2192.
Our own considerations regarding these phenomena aimed for the first time at the integrity of the E-cadherin gene. After RNA extraction, reverse transcription and direct DNA sequencing, the molecular-biological examination of gastric carcinoma tissue revealed defects in the E-cadherin gene. Gastric carcinomas of the diffuse subtype were examined for genetic alterations in a part of the E-cadherin gene (exons 6-10 and 13-16). Loss of exons 8 and 9, partial loss of exon 10, or a point mutation in the region of exon 8 were observed (Becker K-F, Atkinson M J, Reich U, Becker I, Nekarda H, Siewert J R, Hxc3x6fler H: E-cadherin gene mutations provide clues to diffuse type gastric carcinomas. Cancer Res. 1994; 54(14):3845-3852). Tumors of the intestinal subtype showed no mutations leading to structural alterations. Analysis of the found mutations revealed that individual deletions occurred with somewhat higher frequency, but also point mutations or smaller deletions could be observed. Immunohistochemical staining (antibody: HECD-1, Takara Biomedicals, Japan, cf. methodology section) of some of the cases with mutated E-cadherin predominantly showed a transmembrane staining of the tumor cells and also staining of non-tumorous mucosa. We were not able to distinguish whether the labeling of the tumor cells corresponded to the detection of wildtype E-cadherin or mutated E-cadherin. On the one hand, there was the possibility that mutated protein continued to be incorporated into the cellular membrane and that the antibody against E-cadherin (HECD-1) recognizes an epitope apart from the mutated region. A further explanation could be the binding of the antibody to wildtype E-cadherin whichxe2x80x94being generated by the second, not mutated allelexe2x80x94is also expressed in tumor cells. Initially, the fact that some of the tumors showed no staining led us to suggest the presence of further mutations apart from the examined exons 6-10 and 13-16 which might have an influence on the translation or the stability of the protein.
It is an object of the present invention to provide monoclonal antibodies which are specifically suitable for the detection and therapy of gastric carcinomas and in particular diffuse gastric carcinomas.
It is a further object of the present invention to provide methods for the detection and therapy of diffuse gastric carcinomas.
This object is achieved according to the invention by the monoclonal antibodies characterized in more detail in claim 1. Preferred embodiments of the invention follow from the dependent as well as the secondary claims.
The analysis of regions of the E-cadherin gene which have not been examined so far (exons 1-5, 11, and 12), and the sequencing of the cDNA of E-cadherin of ten additional cases of diffuse gastric carcinoma revealed further mutations in diffuse gastric carcinoma (cf. the xe2x80x9cMethodologyxe2x80x9d section). These newly found mutations (Table 2) surprisingly continued to show a typical pattern. In all of the cases, these mutations were either multimers of a base triplet not affecting the reading frame or they were point mutations. By these studies we were able to exclude the suggestion made after immunohistochemical examination that the loss of the immunoreactivity may be caused by translation-disrupting mutations. In parallel to the examination of gastric carcinomas also other epithelial tumors were analysed. None of the cases of mamma carcinoma, oesophagus carcinoma, and large intestinal carcinoma showed patterns corresponding to those of gastric carcinoma.
Additional studies carried out with respect to the characteristics of the mutations as well as sequencing of additional tumors now have led to a principle which has not yet been reported and is unique in the case of carcinomas: the alterations of E-cadherin in diffuse gastric carcinoma are in-frame mutations (and not disruptions of the reading frame). This result is useful for diagnostic and therapeutic purposes, for example the therapeutic use of antibodies, which recognize mutant E-cadherin amino acid sequences.
As far as these therapeutic uses are concerned, the use of radiolabeled MAbs (monoclonal antibodies) in radioimmuno-therapy has been limited up to now because of the lack of tumor-specific antigens. In most cases reported thus far, tumor antigens that serve as targets are not tumor-specific, being overexpressed by tumor cells and also at a lower level by normal cells. Thus far, only one tumor-specific MAb has been reported that recognizes a mutant form of the epidermal growth factor receptor (EGFR vIII) that is found on different tumor types but not on normal human tissue (1). This antibody has been labeled with 125I, 131I, and the xcex1-emitter 211At, and it appears to be a promising candidate for radioimmunotherapy (2, 3).